Assaying glycated proteins is very important in diagnosing and controlling diabetes. Glycated hemoglobin (GHb) that reflects an average blood glucose value in the past about 1 to 2 months, glycated albumin (GA) that reflects an average blood glucose value in the past about two weeks, fructosamine (FRA) which generally indicates glycated proteins exhibiting reduction capability in blood serum, and the like are measured every day. GHb is a glycated product of hemoglobin, i.e., the α-amino group of N-terminal valine on the β-chain of hemoglobin is glycated. GA and FRA are glycated products of albumin and blood serum protein respectively, i.e. the ε-amino group on a lysine residue of albumin or blood serum protein is glycated.
An enzymatic method is given as a simple, easy, and inexpensive method for accurately assaying glycated proteins. Japanese Patent Applications Laid-open No. 6-46846, No. 5-192193, No. 2-195900, and No. 2-195899, and International Patent Application Publication Number WO 98/48043, and WO 97/13872 are given as examples of documents disclosing the enzymatic method.
However, to provide a composition for precisely assaying glycated proteins, it is essential 1) to avoid the effect of globulin components and ascorbic acid and 2) to stabilize protease, at least the enzyme that reacts with glycated amino acids. In addition, in the case where the glycated protein is a glycated albumin, it is important 3) to precisely assay albumin and 4) to avoid the effect of glycated hemoglobin.
1) Conventional Methods for Avoiding the Effect of Globulin Components and Ascorbic Acid
It is known that the amount of globulin proteins of a diabetic changes and affects the value of FRA [Rodrigues, S. et al., Clin. Chem. 35: 134-138 (1989)]. The present inventors have developed a method of selectively inhibiting the action of a protease on globulin components by adding a specific metal ion and a protein A or G to a protease reaction solution (Japanese Patent Application No. 11-231259). Glycated proteins can be assayed without being affected by globulin components using this method of the present invention. As a globulin-selective protease inhibitor used in the method, metals, protein A, and protein G are mentioned. Among the metals specified in this patent application, highly effective metals are heavy metals that may have environmental safety problems. Less effective metals may make a reagent solution turbid if combined with other reagents (or compositions). In addition, protein A and protein G are very expensive reagents.
As a method for selectively adsorbing globulin in blood, a blood-treating agent adsorbing endotoxins and globulin in blood utilizing the principle of chromatography and a vinyl copolymer introduced a ligand having a steroid skeleton is known (Japanese Patent Application Laid-open No. 61-94663). However, the results shown in Table 1 of Examples of Japanese Patent Application Laid-open No. 61-94663 indicate that only α1-globulin and α2-globulin have been confirmed to be adsorbed, but γ-globulin that makes up 70% or more of globulin components was not adsorbed. Supposing that γ-globulin was adsorbed, the capability of the blood-treating agent of inhibiting protease activity on γ-globulin cannot be anticipated.
Occasions of a large amount of ascorbic acid intake as a supplement are increasing in recent years. Clinical samples containing ascorbic acid at a high concentration are also increasing. Ascorbic acid induces a variety of effects on clinical examinations due to the strong reducing action.
As a method for obviating the effects of ascorbic acid, a method of eliminating the ascorbic acid in samples chemically or enzymatically using an ascorbic acid oxidase has been known. When glycated amino acids produced by fragmenting glycated proteins with a protease are assayed using an enzyme which reacts at least with a glycated amino acid, a method of previously eliminating ascorbic acid using an ascorbic acid oxidase (ASOx) at the time of reaction with a protease is preferable in view of a small effect on the coloring system.
As an example of eliminating ascorbic acid in samples using ASOx in the presence of a protease, an experiment of reacting ASOx with a sample solution in a 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid (HEPES) buffer solution at pH 8.0 has been reported (Clinical Chemistry, 27: 99-106, 1998). The report describes that the ascorbic acid treating capability did not change after cold storage for two weeks.
However, the investigation made by the present inventors has revealed that when a HEPES buffer solution at pH 8.0, a protease, and ASOx are present, the ascorbic acid treating capability has been lost almost entirely in one day when stored at 37° C. or in two weeks when stored at 10° C.
2) Prior Art for Stabilizing Proteases and Enzymes Reacting at Least with a Glycated Amino Acid
A solution of protease at an inconceivably high concentration of a level which cannot be seen in another field such as the food industry is used in clinical assay of glycated proteins. Proteases are known to self-digest in an aqueous solution. It is difficult to assume that a protease remains stable in an aqueous solution at such a high concentration. Therefore, proteases used for a composition for assaying glycated proteins have been supplied as a freeze dry product.
There has been no composition for assaying glycated proteins nor a method of assaying glycated proteins in which a protease is stabilized in a liquid state storable for a long period of time. There has also been no composition for assaying glycated proteins nor a method of assaying glycated proteins in which the enzyme reacting at least with a glycated amino acid was stabilized in a liquid state storable for a long period of time.
3) Prior Art Relating to a Method for Precisely Assaying Albumin
Anti-albumin antibody immunization and a dying method using bromocresol green (BCG), bromocresol purple (BCP), or the like are given as the method for assaying albumin. The dying method is widely used in everyday inspections due to the simple procedure and low cost. Although the effect of BCG on globulin component has been confirmed, BCG has disadvantages of low specificity to albumin.
On the other hand, BCP is easily affected by coexisting substances in spite of the high specificity to albumin. In particular, BCP is affected by SH compounds giving rise to a problem of variation in the assay results according to oxidation-reduction conditions of albumin. As a means for solving this problem, a method of reacting BCP in the presence of a protein denaturing agent and/or an SH reagent has been proposed (Japanese Patent Application Laid-open No. 10-232233) However, there have been no examples on the study of the reactivity of BCP to GA and non-glycated albumin (NGA).
4) Prior Art for Avoiding the Effect of Glycated Hemoglobin
As mentioned above, GA is derived from albumin by glycation of the ε-amino group, whereas GHb is obtained by glycating α-amino group of N-terminal valine on the β-chain of hemoglobin. Therefore, in case GA is applied as measuring object it is desirable to determine only amino acids in which the ε-amino group has been glycated.
Although several enzymes exhibiting high specificity to ε-amino group but no action on glycated valine has been known (Japanese Patent Application Laid-open No. 11-243950), none of them are supplied at a sufficiently low cost for the enzymes to be used in practice. Of these enzymes, a fructosyl amino acid oxidase (FOD) derived from Fusarium oxysporm possesses high reactivity and is useful. The inventors have reported separately the gene of FOD (Japanese Patent Application Laid-open No. 10-201473). Although the process using the gene exhibits high productivity and can produce FOD at a low cost, the reactivity with glycated valine of which the α amino group has been glycated confirmed by the inventors does not exhibit satisfactory specificity.